An effective HIV-1 vaccine approach not only will need to induce broadly reactive neutralizing antibody and cellular immune responses at systemic and mucosal sites, but the vaccine also will have to be feasible for distribution to parts of the world that need it most in terms of safety, manufacturing cost, number of inoculations, and ease of administration. The common cold human coronavirus (HCoV) OC43 is attractive as a vaccine vector for HIV, and recent breakthroughs in coronavirus reverse genetics have now made it feasible for their exploitation as vaccine vectors. Coronavirus genomes are the largest RNA genomes in nature (~30kb), and contain multiple genes that are not essential for viral replication, theoretically allowing the insertion of multiple heterologous genes into a single virus with a packaging capacity much larger than most other vector systems. Because HCoV OC43 causes only a mild upper respiratory tract infection, it is anticipated to be relatively safe as a vaccine vector, allowing for replication competent viruses to be used to present heterologous antigen at mucosal surfaces for multiple rounds of vector replication, much like a live attenuated vaccine. Coronavirus genomes also can be engineered such that recombination with naturally circulating strains yields a dead virus. We recently completed the development of a reverse genetics system for HCoV OC43, which now makes it technically feasible to develop a HCoV OC43-based vaccine vector. In this phased innovation R21/R33 project we propose to engineer the HCoV OC43 genome as a multiple heterologous gene expression vector for SIV, which we will assess using the SIVsmE660/macaque model. In the R21 proof-of-concept phase we will: (i) engineer HCoV OC43 to express the SIVsmH4 matrix/capsid (MA/CA) gene from different regions of the vector genome, and (ii) evaluate the systemic, mucosal, and cellular immunogenicity of MA/CA-expressing HCoV OC43 vectors in mice. In the R33 phase we will: (i) characterize infectivity of HCoV OC43 in macaques, (ii) engineer and validate a recombination-resistant HCoV OC43 construct to express up to four SIVsmH4 proteins-Gag, Env, Nef and Vif, and (iii) vaccinate macaques with the multigene HCoV OC43 vector and assess vaccine efficacy by mucosal challenge with SIVsm E660. This project will provide the first critical evaluation of the potential use of common cold human coronaviruses as live mucosal vaccine vectors for HIV. PUBLIC HEALTH RELEVANCE: In the proposed study we will develop human coronaviruses, which cause about one- third of all common colds, as vaccine vectors to deliver HIV antigens. There are several potential attractive features of coronavirus vectors as vaccines for HIV, such as safety, simplicity in manufacturing, the ability to induce immune responses in sites of HIV transmission, and the ability to induce immune responses with a limited number of inoculations. We will evaluate the potential use of coronaviruses as HIV vaccine vectors using the SIV/macaque model.